There is a growing appreciation of aldose reductase "activation" as a potential risk factor in diabetic complications. Activation may be central to why some diabetic patients experience severe tissue degeneration while others with comparable hyperglycemia survive 40 years of disease with little or not complications. This new perspective stems, in part, from our studies of purified bovine kidney aldose reductase showing that the enzyme can exist in either and unactivated or an activated state, with a 17-fold difference in turnover number for the two enzyme forms. Evidence for activation of aldose reductase has been reported in several mammalian species, including human. The hyperglycemia-induced increase in reaction flux through the polyol pathway (aldose reductase-catalyzed NADPH-dependent reduction of D-glucose to D- sorbitol followed by NDA+-dependent reoxidation to D-fructose) has been directly linked to the subsequent development of diabetic complications in general, and of diabetic kidney disease in particular. Activation of aldose reductase can magnify this effect leading to greater tissue damage at a fixed level of hyperglycemia. The design of specific aldose reductase inhibitors (ARI) has thus become a major area of research effort. Yet, results from clinical trials in humans have not been as promising as expected based on the success in animal models. We believe that activation in vivo may be at fault. Activation of bovine kidney aldose reductase in vitro leads to a pronounced change in the potency of some, but not other ARI. Combined with evidence for a difference in ARI binding stoichiometry, these results suggest that ARI can bind to aldose reductase in different modes and/or site(s), and have lead to a proposal for a new ARI classification system based on the sensitivity to the activation state of the enzyme. In this application, we will directly test the hypothesis that aldose reductase activation is a predictor for increased risk of developing diabetic kidney complications in humans. We have developed methods that allow direct quantitation of the amount of aldose reductase protein, the total activity, and the activation state of the enzyme in tissue samples. This method will be used to correlate the extent of enzyme activation with the severity of diabetic kidney disease using tissue from normal and diabetic patients. We ill use conventional and tight-binding kinetic methods and pH studies to compare and contrast the two ARI classes. Computer-aided QSAR studies will then be used to identify and analyze the important structural factors (electrostatic, steric, hydrophobic) that determine the sensitivity of ARI potency to the activation state of the enzyme. Using the protein chemistry knowledge base developed during the determination of the primary sequence for bovine lens aldose reductase, we will investigate the molecular basis for enzyme activation, focusing on the role of sulfhydryl oxidation.